Biochemistry and biomechanics of healing tendon: Part I. effects of rigid plaster casts and functional casts. Med. Sci. Sports Exerc., Vol. 30, No. 6, pp. 788-793, 1998.
Purpose: Traditional treatment of surgically repaired Achilles tendons includes complete immobilization of the joint in rigid casts for 6 to 8 wk. We tested the use of functional polyurethane casts as an alternative to rigid plaster casts after experimental tenotomy and repair of the rabbit Achilles tendon.
Methods: After repair the limbs of 15 experimental rabbits were immobilized in a functional polyurethane cast for 15 d, while those of 14 controls were immobilized in traditional rigid plaster casts for the same period.
Results: Functional casting resulted in a 60% increase in total collagen in the neotendon compared with that in rigid casting (P < 0.05). Mature collagen cross-links declined 8% in the tendons with functional casts. The biomechanical parameters of the tendons changed with functional casting, showing a 20% increase in maximum load and 21% increase in maximum stress.
Conclusions: These changes were noted without any cases of tendon re-rupture in either type of cast. Thus, functional casting following surgery of Achilles tendons appears to improve healing without significant risks of re-rupture.
Department of Physical Therapy Education, University of Kansas Medical Center, Kansas City, KS 66160-7601
Submitted for publication January 1997.
Accepted for publication November 1997.
This work was supported by the Department of Veteran Affairs, Rehabilitation, Research, and Development Program, NIDRR (H133G20220) and NIH-NICHD.
Address for correspondence: Dr. Chukuka S. Enwemeka, Department of Physical Therapy Education, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7601. E-mail: firstname.lastname@example.org.
Injuries to tendons are commonly seen in orthopedic practice and have increased in incidence recently (26). The injury may be a result of mechanical overload or of repetitive trauma. About 32% of all surgery relating to overuse involves tendons. The most common site of injury is at the Achilles tendon (31). Additionally, tendon surgery is performed as a corrective measure for patients with neurological deficits resulting in abnormal tone that pulls the extremity into undesirable positions. Children with cerebral palsy frequently undergo corrective orthopeadic surgery that includes the release, transfer, or lengthening of the Achilles tendon (9).
Whether the insult to the tendon was a result of injury or corrective surgery, rehabilitation following tendon surgery presents a unique set of clinical problems because of the extended time that the tendon is immobilized (20,23). Some authors suggest a minimum of 8 wk of immobilization following Achilles tendon injury (19). Immobilization has been documented to produce a variety of complications including muscle atrophy (3,6,11), joint stiffness (22,39), atrophy and ulceration of joint cartilage (1), osteoarthritis (22,38,1,21,32), skin necrosis, infection (5,24), tendocutaneous adhesion (4,10), and thrombophlebitis (10,29). Other studies illustrate that immobilization negatively alters the ultrastructure (14), biochemistry (25,33), and biomechanics of the tendon (2,13,30). Human studies have shown that 6 months after surgery the Achilles tendon has not recovered concentric or eccentric plantar flexion muscle strength compared with noninjured tendons (34).
To minimize the detrimental effects of rigid immobilization, functional ankle casts that allow some degree of movement have been tested clinically (7,8,37). The casts allow the ankle to flex to 20% plantar flexion (8) or to neutral ankle position (7), with toe-touch to full weight bearing allowed in both designs (37). Patients treated with movable casts or orthoses have shown equal or improved recovery with few complications compared with patients treated with fixed casting methods (7,8,37). However, it is difficult to draw conclusions because the studies lack controls (37). In spite of these encouraging reports, the practice of early weight bearing and functional immobilization following Achilles tendon surgery has not been adapted since controlled studies documenting the objective effects of functional casting on tendon healing remain lacking.
The purpose of this study was to compare the effects of rigid cast immobilization and functional cast immobilization on the healing process of experimentally tenotomized and repaired rabbit Achilles tendons. Specifically, our aims were to compare the biochemical and biomechanical indices of healing in both groups of tendons. We have used the rabbit Achilles tendon model to study the healing process of connective tissue extensively and have reported previously that 15 d following tenotomy and repair, the biomechanical and biochemical changes of interest have peaked. Thus, this work studied the effects of rigid cast immobilization versus functional casting on rabbit Achilles tendons 15 d after repair.
Animal protocols. The methods for animal care and surgical protocols have been described (13). The full protocol was reviewed and approved by the institutional animal care and use committee. Briefly, 29 rabbits age 10-12 wk were used in the study. Experimental tenotomy was performed as described previously (13). Rabbits were treated with intra-muscular injections of 3 mg·kg−1 body weight Xylazene and 35 mg·kg−1 body weight Ketamine. Subsequently, the skin overlying the right Achilles tendon was shaved, scrubbed, and anesthetized locally with 2 mg·kg−1 body weight lidocaine. The right Achilles tendon of each rabbit was then tenotomized with a sharp transection approximately 1.5 cm above the calcaneal attachment. The severed ends were then reapproximated and sutured. Following experimental tenotomy and surgical repair of the right Achilles tendon of each rabbit, either a rigid plaster of Paris cast or a functional polyurethane cast was used to immobilize the repaired tendon. To promote rapid recovery from anesthesia, an injection of 0.2 mg·kg−1 body weight Yohimbine was given. Rabbits were kept warm in an oxygen chamber until they regained consciousness.
Casting procedure. Before full recovery from anesthesia, each surgically repaired tendon of 15 rabbits was immobilized in a plaster of Paris splint using a technique developed in our previous studies (13,15-18). In this method of casting six layers of 4-inch plaster, trimmed to the shape of the limb, were molded into a rigid frame covering the anterior surface of the limb. The rigid frame was then anchored to the limb using four rings of 2-inchwide plaster. To prevent gnawing of the plaster by the rabbits, the plaster was protected with an outer shell of fiberglass.
The repaired tendons of the remaining 14 rabbits were immobilized in functional polyurethane (Smith and Nephew Rolyan, Inc.) casts before full recovery from anesthesia. Frames of polyurethane casts were cut to the shape of the limb and preformed in warm water. The hardened frame was anchored snugly to the limb with fiber adhesive tape permitting limited use of the limb and minimal restriction of blood flow. The limb was also positioned in full plantar flexion and 90% knee flexion for all casts. After the material set, adhesive material was attached to each cast above and below the knee. At the time of casting fine gauze was inserted in each cast to protect the skin. The basic designs of both casts were identical with full plantar flexion and 90° flexion at the knee.
Following tenotomy and casting, the animals were observed during recovery and allowed food and water ad libitum. Animals were allowed free movement about the cage for the next 14 d. Fifteen days following surgery, casts were removed, and animals were weighed and euthanized with an overdose of sodium pentabarbitol. Tendons were removed, weighed, and stored for biomechanical and/or biochemical assays.
Biochemistry. Biochemical assays were performed using techniques described previously (35). Briefly, total collagen within the tendon sample was determined by measuring the concentration of hydroxyproline using our published procedures (35). The dried tissue specimens were homogenized in cold saline, hydrolyzed in alkali, and oxidized with chloramine-T. The chromophore was developed with the addition of Ehrlich's aldehyde and the absorbance was measured at a wavelength of 550 nm. Concentrations of hydroxyproline were deduced from a standard calibration curve, and collagen amounts were calculated based on hydroxyproline comprising 14% of the total amino acids of collagen.
Cross-links were measured with high performance liquid chromatography (HPLC, Shimadzu) using reverse phase columns equilibrated with 21% acetonitrile in water containing 0.01 M n-heptafluorobutyric acid. Hydroxypyridinium cross-links were resolved using a linear gradient from 21% to 25% acetonitrile. Pyrodoxamine was used as the standard to calculate the concentration of cross-links.
Biomechanics. Biomechanical measurements were taken using techniques described previously (13). Briefly, the cross-sectional area of each tendon at the region of the neotendon (site of the tenotomy) was calculated by measuring the diameter to the nearest 0.1 mm using a customized caliper. Each excised tendon was clamped at the tendonous junction and above the calcaneal insertion using modified pneumatic clamps of the Instron (Canton, MA) materials testing device. Each tendon was pulled to rupture, which always occurred at the site of the tenotomy. We have described the customized clamping method previously (18), which allows for successful gripping of the tendon with undetectable slippage of the tendon and clamp. Using a 500-N load cell, the tendons were each pulled to rupture at a speed of 250 mm·min−1. The biomechanical parameters of each tendon were computed, including load (measured to the nearest 0.01 N), displacement (measured to the nearest 0.001 mm), stress, strain, Young's modulus of elasticity, and energy absorbed (Fig. 1).
Data analysis. Multivariant analyses of variance (MANOVA) tests were used to compare the biomechanical and biochemical characteristics of the two groups of tendons. Reported data represents the mean ± SE.
While the total length and shape of the two casts were identical, the plaster of Paris cast weighed two times more than the polyurethane cast (plaster: 211.7 ± 6.3 g; polyurethane casts: 90.2 ± 5.0 g; N = 6/group). Both casts held the ankle in an equinus position of plantar flexion. The functional cast allowed movement at the ankle joint of 15% from full plantar flexion. In addition, the functional cast allowed 5% of ankle inversion from a neutral position. In contrast, the rigid cast allowed no movement at the ankle. All animals tolerated the casting protocols equally well, and there was no re-rupture of the tendon in any of the animals tested.
The animals in rigid plaster of Paris casts lost a significant amount of weight during the 15 d of the experiment, even though both groups were allowed food and water ad libitum. There was a 6.3% drop in weight in the rigidly-casted animals but only a 0.4% loss in weight in the functionally-casted rabbits. In both cases rabbits were weighed without the casts; thus, the casting material itself did not influence the animal weight values. Observation of the rabbits during this period indicated that the rigidly-casted limbs were typically held abducted from the side of the animal in a position that did not allow weight bearing, while flexibly-casted rabbits appeared more mobile, although these observations were not quantified.
Biochemical assays. We compared the biochemical properties of the tendons in functional or rigid casts (using six samples per group). Tendons in functional casts had 60% more total collagen with 440.1 ± 27.3 mg·mg−1 dry tissue than those in rigid casts; 278.4 ± 25.9 mg·mg−1 dry tissue (Fig. 2). Tendons with more total collagen also had fewer total cross-links 271.9 ± 21.8 and 294.4 ± 30.3 pM·mg−1 dry tissue for polyurethane- and plaster-casted tendons, respectively (Fig. 2). Thus, the use of polyurethane casting material significantly increased (P < 0.05) the amount of total collagen in the healing tendons, but with no significant change in mature cross-links.
Biomechanical assays. The mean cross-sectional area of the tendon at the tenotomy site was not significantly different with 63.7 ± 3.5 mm2 for tendons in rigid casts and 61.9 ± 3.9 mm2 for those in functional casts (P > 0.05). When tendons were pulled to rupture, all tendons from limbs in either functional or rigid casts ruptured at the site of the tenotomy. There were no significant changes in maximum load; 145.8 ± 10.2 N for functional casts versus 121.3 ± 8.9 N for the rigidly-casted group (P > 0.05). For all biomechanical tests N = 15 for rigidly-casted limbs, and N = 14 for functionally-casted limbs. Maximum stress as a result of functional casting was 2.46 ± 0.20 MPa compared with 2.03 ± 0.21 MPa for the rigid cast (P > 0.05; Fig. 3).
The displacement (distance that tissue stretched) was not different between the two groups (maximum displacement was 7.32 ± 0.59 mm for polyurethane and 7.26 ± 0.57 mm for plaster). Strain is a measurement of tissue elongation divided by the original length of tendon section tested (displacement/gauge length). For these experiments strain indicates the displacement of the whole tendon. This normalized parameter takes into account the different initial lengths of the tendons resulting in percent change in displacement of the tendon as it is pulled to rupture. While the majority of the strain occurred at the site of tenotomy, the exact percentage of strain that developed in other portions of the tendon could not be identified. Strain at maximum load did not change significantly with the functional cast as illustrated in Figure 4A (52 ± 3% for the functional group vs 42 ± 4% for the rigidly casted group; P > 0.05).
Young's modulus of elasticity was 9.2 ± 0.8 MPa for the polyurethane-casted group compared with 10.0 ± 1.1 MPa for plaster (no significance, P > 0.05; Fig. 4B). The energy absorbed by both groups of tendons to maximum load was less with functional casts, but not statistically significant (P > 0.05; Fig. 4).
There is much controversy surrounding the postsurgical management of Achilles tendons (38), but the majority of clinicians believe that rigid casting is necessary to immobilize the limb (8,19). Conversely, there is little question concerning the positive benefits of early mobility and weight bearing following a lower extremity injury. These activities promote normal healing (28), inhibit muscle atrophy (13), and increase blood supply (36). Thus, the findings of this study, that functional casting during the early healing phase of Achilles tendon repair improved some of the biochemical and biomechanical properties of the tendon without re-rupture, has important clinical relevance.
Tendons from limbs immobilized in the functional cast improved in the maximum load they withstood by 20%, and maximum stress by 21% in this study, although these changes were not statistically significant. Since only one time point was measured in these studies (15 d postsurgery), it is possible that other biomechanical and biochemical changes occurred during the course of the healing process that were not present at this time point. Results of a recent study (27) on the effects of motion and load on injured tendons is consistent with our findings. Kubota et al. (27) found that the application of both movement at the joint and load increased the load-at-failure values over control chicken tendons four weeks following injury.
In previous studies using rigidly-casted procedures, we showed that tendons receiving treatment with laser stimulation had dramatically greater maximum load and greater energy absorption capacity than control tendons (12). He:Ne laser treatment increased maximum load (tensile strength) 42% with the application of the laser to rigidly-casted lower extremities. In contrast, the improvement in maximum load when comparing functional casting with rigid casting was only 20%. In our original publication, laser photostimulation alone improved the energy absorption by 33% (12), while functional casting caused the energy the tendon could absorb to increase by only 8%. Similarly maximal stress improved by over 100% with the application of the He:Ne laser while in this study we report an increase in stress of 21%. Thus, functional casting improved some of the biomechanical properties of the tendon, but not to the magnitude of laser treatment of the tendon. Neither optimal casting techniques or laser treatment have been shown to improve the biomechanical properties of the tendon to a level equal to noninjured tendons. It is possible that the combination of optimal immobilization procedures like functional casting along with laser stimulation would stimulate tendon healing further.
The results of experiments reported here indicate that immobilization in functional casts enhances some indices of rabbit tendon healing at both the biochemical and biomechanical levels. This conclusion supports the few published clinical trials using functional casting for repaired human Achilles tendons. Those studies could not evaluate the biochemistry or biomechanics of the healed tendons, but showed equal or improved recovery with the functional casts (7,8,37). Our studies indicate that positive changes occur in the tendon with functional casting at both the molecular and whole-tissue levels, thus suggesting a mechanism for the clinical observations.
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